The goal of these studies is to determine the required biochemical events occurring between the interaction of tumor promoters with the plasma membrane and the activation of effectors of neoplastic transformation. Candidate second messengers include protein phosphorylation, reactive oxygen generation, and calcium mobilization. Recent evidence indicates that the free radical superoxide anion, but not hydrogen peroxide or singlet oxygen, appears to be an essential mediator of neoplastic transformation by tumor-promoting phorbol esters in JB6 mouse epidermal cells. The superoxide anion is apparently critical during the first two hours after the phorbol ester interaction with its receptor. The enzyme, superoxide dismutase (SOD), inhibits promotion of transformation by TPA only when added to cells up to two hours after TPA. We have previously reported that the major epidermal cell surface ganglioside, trisialoganglioside (GT), shows substantially decreased net synthesis after tumor promoter exposure in promotion-sensitive (P+), but not in promotion-resistant (P-), JB6 cells. This observation led to the hypothesis that GT is an oxidative target of oxygen radicals generated after TPA exposure, and that this oxidation leads to decreased GT synthesis and promotion of transformation. Predictions of this hypothesis have been confirmed in a recent article demonstrating that sodium metaperiodate (NaI04), a known oxidizer of cellular GT sialic acid, produced both decreased GT synthesis and promotion of transformation. Recent evidence indicates that promotion of transformation in JB6 cells requires both extracellular calcium and calcium mobilization from bound pools. It is postulated that certain calcium-dependent enzymes, including the phorbol ester receptor, protein kinase C, play a regulatory role in tumor promotion. The proposed course will be concerned with : (1) determining the promotion-relevant, membrane lipid targets of reactive oxygen generated by phorbol esters, (2) characterizing the decrease in SOC by tumor promoters, (3) elucidating the calcium-dependent processes that regulate tumor promotion, and (4) understanding how these signal transduction events regulate the expression of pro genes.